week 8 part 1 Flashcards
What is Hemophilia?
A severe bleeding disorder
A condition which has deficiency of factor 8
Gives rise to Haemophila A and B
Who was the most famous carrier for the defective gene for Haemophila B
Queen Victoria
What does bleeding disorder in the absence of any therapy mean?
Bleed for 5-6 weeks and need to be in hospital with clamp
people used snake venoms before they worked out what to do therapeutically
What were some of the consequences as a result of missed opportunities?
- prolonged medicalization
- Missing school
- poorly educated
What is common in the absence of treatment?
very severe joint damage
avoid bleeding of the head
What is a guranteed problem as a result of escape head bleed?
musculoskeletal damage
What has been damaged in haemohpila?
femur and tibia
shows muscle wasting
What did 70% of the world’s haemophiliacs have?
Limited therapy options
What is haemophilia?
A global disease
not pre-directional to any ethic group
What does the UK realise for haemophilia?
Clotting factors have occurred in the plasma
Fractionate blood to derive a specific plasma factor
What is a real drive in Bangladesh?
Reduce risk from therapeutic agents
What was thought to be in the factors of haemophila?
- Variant CJD
- Mad cow disease
- Prion agents
what is the mainstay of treatment for haemophila A and B?
Fresh frozen plasma (FFP)
Why were children usually hospitalized ?
Treatment of bleeding into a knee, an elbow or other joint
What is recombinant clotting technology?
synthetic clotting factors that has not been derived from a donor
What do patients with severe hemophila produce?
less than 1% of the normal amount of the affected clotting factor
dependent on factor from intravenous infusions to treat or prevent bleeding episodes
What was discovered in the 1980’s?
human blood, plasma, and plasma-derived products were discovered to be transmitting potentially deadly blood-borne viruses, including hepatitis viruses and HIV
what did manufacturers of plasma-derived clotting factor concentrates attempted to do?
kill these viruses with dry heat, solvent-detergent treatment, and pasteurization, with varying degrees of succes
What happened during the mid-1980’s?
the genetic sequence of FVIII gene was achieved to produce recombinant factor VIII
What was the advantage of recombinant FVIII
Did not require any type of plasma for its production, first patient treated was reported in 1987
no reports of viral transmission linked to the use of rFVIII in the developed world
How is haemophila B currently treated ?
Frequent injections of a concentrated form of FIX protein
What is the gold standard treatment for the severe disease?
- prophylaxis
with infusions twice a week for haemophila A
and once every 1 to 2 weeks for hemophila B
What was the breakthrough in modern era gene therapy?
used wild-type FIX in AAV8 vector containing a liver-specific promoter enabling peripheral administration
What are AAV vectors?
episomal and do not integrate, so some loss of efficacy would be expected as the transduced hepatocytes turn over
What didnt the participants in any of the gene therapy trial produce?
inhibitors
What are the normal factors between?
50-150
What is Adeno-associated virus (AAV)?
protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb).
dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate.
What can gene therapy vectors using AAV infect?
both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell
What are the characteristics of gene therapy for Hemophila?
- latitude in the choice of the target tissue
- Biologically active clotting factors can be synthesized in a range of cell types, and will be effective so long as the gene product reaches the circulation
- wide therapeutic window.
What are AAVs?
among the smallest of the animal DNA viruses, with a ~5-kb genome packaged within a ~26-nm non-enveloped, icosahedral capsid
What does typical AAV genome contain?
Two protein-encoding genes termed rep and cap that, respectively, encode nonstructural proteins essential for viral genome replication and structural proteins that form the viral capsid.
What does the AAV genome consist of?
rep and cap genes responsible for the viral life cycle and the formation of the capsid shell surrounding the genome
What is Gene delivery systems categorised as?
- viral based
- Non-viral based
- combined hybrid systems
what does viral-mediated delivery system consist of
Viruses that are modified to be replication-deficient, but which can deliver DNA for expression
What are used as Viral gene-delivery vectors?
- Adenovirus
- Retroviruses
- Lentiviruses
What are the most common physical methods?
- Microinjection
- Electroporation
- Ultrasound
- Gene gun
- Hydrodynamic applications
What is the main goal of gene therapy?
treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels.
What does episomal vector system have the potential to avoid?
potential risk of causing insertional mutagenesis
What does episomal vector system behave as?
separate extrachromosomal elements in the nucleus of a target cell
Why did Adenovirus in 1990’s kill somone in early trials?
There was such a profound immunological reaction when it was infused
What are some of the examples of moving DNA in viruses?
- Retrovirus
- Adenovirus
- Adeno-associated virus
- Lentivirus (modified HIV)
What are the synthetic/non-viral way of moving DNA?
- direct gene injection
- Liposomes/lipids
- Receptor mediated endocytosis
What are the challenges in gene therapy?
It is very hard to introduce new genes into cells of the body and keep them working
What does gene therapy approached include?
- Gene addition
- Gene knockdown
- Gene alteration/correction
What does the Gene transfer vectors involve?
the use of non-viral DNA plasmids or are derived from the modification of natural viruses.
What are two of the most important goals of gene therapy research?
- improved vectors
2. better understanding of vector-host interactions
What is adeno associated virus a part of?
Adeno infection
Where was initial studies performed?
- wild type mice
2. Hemophilia A dog model
What were the wild type mice not able to provide?
Information about homeostasis
What were the dog model limited by?
- expense
2. long generation time of the dog
what was the attempts to use hemophilia B mice to model?
an apparent CD8+ cytotoxic T-lymphocyte (CTL) response that was seen in a human clinical trial of FIX gene therapy
What can be mapped of the mice model?
Mouse strain-specific class MHC I responses that direct CD8+ T cell responses to FIX epitopes on FIX
what doesnt the hemophila mouse model predict?
occurrence of the CTL response
What was the CTL response mediated by?
preexisting memory CD8+ T cells: such cells will be present in humans as the natural host of wild type AAV, but mice do not naturally host this primate virus.
What do both adenovirus and adeno-associated virus share?
- broad host range
2. ability to infect both proliferating and quiescent cells
what can adenovirus accomodate?
- larger inserts
- mediate transient but high levels of protein expression
- easily produced at high titers
what are the features of adeno-associated virus?
- lack of pathogenicity
- added safety due to its replication defectiveness
- ability to mediate long-term expression in a variety of tissues
Annual use of normal medicine
infuse IV, drops profoundly
what is SPK-9001?
experimental treatment for hemophila B that is currently being investigated in an ongoing phase 1/2 trial as a potential single dose therapy
when do you not give gene therapy?
someone who has pre-existing immunity
What is SPK-9001?
investigational vector that contains a bio-engineered adeno-associated virus (AAV) capsid and a codon-optimized, high-activity human factor IX gene enabling endogenous production of factor IX
For the safety of SPK-9001, what did 2/9 patients have?
asymptomatic, transient elevation in liver enzymes
what did levels of FIX:C achieved by SPK-9001 permit?
termination of prophylaxis, prevention of bleeding, and nearly complete cessation of factor use
what is the lowest dose currently reported in hemophilia gene transfer trials?
A vector dose of 5x1011 vg/kg
What does the absence of any observed CD8+ T cell immune response support?
The hypothesis that lowering the dose can reduce or eliminate the risk of a capsid-specific immune response and maximize efficacy.
What does the preliminary data suggest about SPK-9001 safety?
Sustained elevation in FIX:C levels sufficient to prevent spontaneous hemarthroses without the need for factor consumption or immunosuppression
What happens if someone is born with non-severe hemophilia but their level of expression is above 5%?
they bleed very little
The more expression you are born with
the less likely u are to have spontaneous bleeds
What do all AAV have?
have different serotype conversion rate in the population
What are the common properties of AAV serotypes?
- non-enveloped, single-stranded DNA parvoviruses, 25 nm in diameter
- genome size and organization
- inverted terminal repeats share structure and function
- rAAV retain abilities to package and transform
What are the differences of AAV serotypes?
- variable tropism
- amino acid identity in the capsid protein AAV serotypes 1–9 ~45%
- most divergent AAV4 / AAV5
- Variable population immunity
What has been generated using AAV2?
he greatest amount of in vivo transgene expression
What is BioMarin?
developing gene therapy using an AAV-factor VIII vector, valoctocogene roxaparvovec, an investigational drug for the treatment of hemophilia A
What did mouse models of hemophilia A, valoctocogene roxaparvovec restore?
factor VIII plasma concentrations to levels projected to be adequate for normal clotting in humans
What did BMN 270 injected into hemophilic mice result in?
dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss
Why did Hemophilia A gene therapy has proved more challenging?
- size of the FVIII gene
2. the poor cellular expression efficiency of full-length FVIII protein.
What is BMN270?
a replication-incompetent AAV5 vector containing a 4.97 kb genome encoding a codon-optimized B domain-deleted (BDD) human coagulation FVIII under the control of a small, liver-specific promoter (HLP)
What is the inclusion criteria of BMN270?
- Look at adult treated with medicine over many years, who have regular treatment or be on demand
- 18 years or older
- severe haemophilia A <1 IU/dL
- > 150 exposure days (EDs)
- > 12 bleeding episodes in 12 months if on-demand
- Prophylaxis pts can be included• No history of inhibitor
- Double barrier contraception for at least 6 months post-treatment
What is the exclusion criteria of BMN270?
- Pre-existing immunity to the AAV capsid – excluded from the trials
- AAV transduction inhibition and AAV total antibodies
- Active infection or any immunosuppressive disorder
- HIV positive, HCV RNA positive, HBsAg positive
- Significant liver dysfunction >3x ULN, significant fibrosis of 3+ rated on a scale of 0-4, liver cirrhosis
- Any other bleeding disorder
- Platelet count of < 100 x 10x9
- Creatinine ≥ 1.5 mg/dL
- Treatment with any IP within 30 days
- Receipt of any vector or gene transfer agent.
- Usual others
What are the outcome for hemophila?
- 5 year follow up
- primary
- Treatment related adverse events
- Expression FVIII (activity assays) - Secondary
- Immune response - Cytotoxic T lymphocytes reaction
- Quantity of FVIII replacement therapy
- number of bleeding episodes requiring treatment
What do guys taking HIV medication have?
Some degree of liver toxicity
What is Valoctocogene roxaparvovec?
AAV serotype 5 vector containing a codon-optimized expression cassette for the SQ variant of B-domain–deleted human factor VIII.
SAFETY OF AAV5-HFVIII-SQ INFUSION
The adverse events that were reported in at least three of the nine participants were an increased alanine aminotransferase level (in seven), arthralgia (in six), back pain (in four), an increased aspartate aminotransferase level (in three), fatigue (in three), and productive cough (in three)
What is the expectation for factor 9?
30-50%
